Immunochromatographic methods have been used as diagnostic means for determining the presence, or absence, of substances of interest or of clinical significance. Generally, these substances are referred to as "analytes" which can consist of antibodies, antigens or other ligands. By taking advantage of the highly specific affinity of known antibodies for specific analytes, immunochromatography is a highly sensitive method for determining the presence or absence of an analyte. Preferably, such methods are simple and easy to judge so that they can be utilized by a broad group of persons, including those with little or no technical expertise.
These methods rely on certain basic known principles. A "first" antibody which binds with specificity to the analyte is affixed near the center of a typically rectangular chromatography medium, e.g. filter paper. A "second" antibody, namely an antibody different from the first antibody yet which also binds with specificity to the analyte, is prepared separately and bound to a detectable marker substance to prepare a marker--second antibody complex. When this second antibody is mixed with a liquid specimen thought to contain a particular analyte, a conjugate of (marker)-(second antibody)-(analyte) is formed. If a liquid specimen containing this conjugate is placed in contact with the edge of the chromatography medium and developed, the conjugate will move gradually in the direction of development and reach the affixed first antibody, thus forming a complex of (marker)-(second antibody)-(analyte)-(first antibody). (In this specification and the following claims, the term "developer" refers to the solvent or moving phase. The direction of development is the direction of the movement of the moving phase.) Because the marker is detectable, the presence of the (marker)-(second antibody)-(analyte)-(first antibody) can be detected. In certain systems, the marker can be detected by the naked eye, i.e. by means of color contrasting with the chromatographic medium. Therefore, a colored mark or the like will be left by the marker at the site at which the first antibody was affixed, (said affixation typically done in the form of a predetermined geometric symbol) and thereby it is possible to easily confirm the presence (or absence) of the analyte.
At present, many such in vitro diagnostic kits based on immunochromatography are known and available commercially, and in most of these, the method of determining the result is based on detecting the presence or absence of a spot or line at the site of the first antibody. When results are determined in this way, it is possible that the positive or negative result may be ambiguous depending on the intensity of the tinting. To solve this problem, a (+) or (-) indicator is used to simplify the judging of results by the user has been proposed, for example, in Japanese Patent Application Public Disclosure No. Sho 64-32169.
In this method, a symbol (+) is placed on the chromatography medium and the first antibody is bound to one line, e.g. the vertical line, of the symbol, while a sample of the analyte or antibody to the second antibody is bound to the horizontal line. If the liquid specimen does not contain the analyte, a complex of (marker)-(second antibody)-(analyte) or (marker)-(second antibody)-(antibody to the second antibody) is formed only on the horizontal line, and the symbol (-) appears as the result. The appearance of this symbol acts as a positive control. If the analyte specimen does contain the analyte, one of the aforesaid complexes will be formed on the horizontal line of the (+) symbol, while a complex of (marker)-(second antibody)-(analyte)-(first antibody) is formed on the vertical line, so the symbol (+) appears as the result. With this method, even in the event that the sample of the analyte and/or the second antibody bound to the marker affixed to the chromatography medium were to become inactive for some reason, or if a major error in handling would occur, the horizontal line will not appear, so it is possible to determine whether the diagnostic test was performed correctly or not. In this manner, this (+) or (-) indicator method has a great advantage in that the validity of the diagnostic test can be verified. Other predetermined geometric symbols can also be used.
However, this method has a major drawback in that it is difficult to get a clear color indication in any line drawn parallel or substantially parallel to the direction of development, for example, the vertical line of the (+) symbol, or portions of "Y" or "N" symbols for the following reason.
If the liquid specimen or solvent potentially containing the analyte having the conjugate of (marker)-(second antibody)-(analyte) reaches the lower edge of the vertical line of the (+) symbol or the vertical lines of "Y" or ,"N" symbols, it reacts with the first antibody forming the complex of (marker)-(second antibody)-(analyte)-(first antibody). However, the complex formed on the lower portion of these lines impedes the further migration of the solvent so the liquid specimen does not come into contact with the portion of the affixed first antibody on the upper part or downstream portion of the vertical indicator lines. As is evident from the FIGS. 1-5, this may result in only small portions of the vertical lines positioned below the other indicator lines reacting or only the top edge and bottom edge of the vertical line reacting, so true (+), "Y" or "N" symbols are not formed correctly. This result occurs if different geometric symbols are used, i.e. only a partial development of the symbol is effected because of the blocking effect of the analyte-marker-second antibody-first antibody complex.
For these reasons, the diagnostic kits presently sold based on the (+) or (-) indicator method must be sold with instructions which indicate that even an incomplete (+) symbol with a missing upper edge or central portion should be considered to be positive. This leads to some confusion, particularly among relatively unskilled technicians or totally untrained consumers with respect to "interpreting" the results, and leads itself to possible mistakes.
In order to overcome the above drawbacks, the following improvements have been proposed.
(1) The line parallel to the direction of development (the vertical line) is pre-printed with the same color as the marker, so that a "+" is indicated when a reaction occurs in the line perpendicular to the direction of development (the horizontal line). An example of this is the commercially available C.A.R.D..+-..RTM.O.S.TM. kit made by Pacific Biotech, Inc.
(2) In another method, the horizontal and vertical lines are made to be oblique with respect to the direction of development. An example is the commercially available TESTPACK+Plus.TM. kit manufactured by Abbott Laboratories, Inc., Abbott Park, Ill.
However, these methods have various drawbacks and are not satisfactory. With method (1), the means of verifying the validity of the diagnostic test (the positive control), namely the method of binding a sample of the analyte to the horizontal line, cannot be employed, so the reliability of the diagnostic test cannot be guaranteed. With method (2), the reaction merely occurs in a skewed "V" shape, so the above drawbacks are not overcome and a true (+) symbol will still not be formed correctly and completely. Therefore, a method which ensures the complete development of the pre-determined symbol is desirable, particularly because complete development will greatly improve the reliability and accuracy of the method.
In light of the above situation, the inventor of this invention made the surprising discovery--as a result of varied efforts at development of a method whereby indicator symbols (a "+", "-", "Y", "N" or some other predetermined symbol) will appear substantially completely and clearly--that by removing a portion of the chromatographic medium, or otherwise partially blocking or obstructing the path of the solvent's movement downstream, complete reaction of the predetermined geometric symbol will occur. As a result of further research, the inventor confirmed that in addition to the widely used (+) or (-) indicators other symbols, e.g. the letters "Y" and "N" which indicate YES or NO, can be utilized with similar clarity.